ES2368298A1 - Method for the delivery of oligonucleotides - Google Patents

Abstract

Method for the administration of oligonucleotides. The present invention deals with a method to obtain formulations that improve the binding capacity of pRNAs to plasma components, which favor the transport of pRNA in the blood, which increase the passage of pRNA through cell membrane, and thus increase the inhibitory activity of pRNAs. The invention describes a process for obtaining a formulation comprising pRNA associated with plasma components, characterized in that the process comprises a stage in which the plasma components are dispersed in an aqueous medium.

Description

Method for the administration of oligonucleotides

The present invention relates to a method for the manufacture of compositions comprising pRNA associated with plasma components. Therefore it belongs to field of biotechnology technique.

Prior art

RNA interference (RNAi) is an important mechanism of gene regulation that can be used for specific gene silencing. The process is initiated by double-stranded RNAs that are called pRNAs, small interfering RNAs (siRNAs). The pRNAs, complementary to a particular messenger RNA, bind to a protein complex known as RISC. The complex formed by the union of the antisense or guide chain with RISC catalyzes the efficient degradation of a specific messenger RNA, causing a decrease in the Diana protein. During the last years, numerous studies have been carried out in order to use this phenomenon of natural gene regulation as the basis for a new therapy aimed at the specific reduction of a previously determined protein. Thus, such therapy could be used for the treatment of diseases known to be caused by overexpression of genes such as cancer or inflammatory diseases [D. Brumcot et al. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nature Chemical Biology 2 (2006), pages 711-719; A. de Fougerolles et al. RNA interference in vivo : toward synthetic small inhibitory RNA-based therapeutics Methods in Enzymology 392 (2005), pages 278-296; C. Chakraborty Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Current Drug Targets 8 (2007) pages.
469-482].

In vivo cellular administration of pRNAs is perhaps one of the most important problems for the development of an efficient therapeutic drug based on RNA interference. [D. Brumcot et al. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nature Chemical Biology 2 (2006), pages 711-719; A. de Fougerolles et al. RNA interference in vivo : toward synthetic small inhibitory RNA-based therapeutics Methods in Enzymology 392 (2005), pages 278-296; C. Chakraborty Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Current Drug Targets 8 (2007) pages. 469-482]. The pRNAs, which are negatively charged hydrophilic molecules, do not, by themselves, have the ability to distribute themselves in the blood and internalize, or penetrate into the cell interior, through the cell membrane that is hydrophobic. To solve this problem, several strategies such as the use of nanoparticles or the use of liposomes have been described [J. Soutschek et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, (2004) pages 173-178; DH Kim et al. Strategies for silencing human disease using RNA interference. Nature Reviews 8 (2007), pages 173-184]. These vehicles can have a double function: to be cellular permeabilizing agents and, in turn, to be protective agents of pRNAs, since naked pRNAs are rapidly degraded by serum RNases. For this reason, a large number of modified pRNAs have been designed and synthesized to take advantage of the binding of modified pRNAs to specific components of the serum (such as albumin, HDL and LDL) and, thus, assist in the distribution of pRNA molecules in the body.

Description of the invention

The present invention is about a method for obtain formulations that improve the binding capacity of pRNAs to plasma components, which favor the transport of pRNA in the blood, which increase the passage of the pRNA through the cell membrane, and thus, increase the inhibitory activity of PARNI. The formulations obtained by the process of the invention clearly improve the entry of pRNAs into cell cultures and the biodistribution of pRNA in tissues. This action is produced by binding of the pRNA to the plasma components, such as the saturable sites of plasma proteins like the albumin, or glycerides such as HDL or LDL, that allow the pRNAI reach the different tissues, remain and be detected even after 4 hours of intravenous administration of pRNA in mice.

Therefore, a first aspect of the invention is a process for obtaining a formulation comprising pRNA associated with plasma components, characterized in that the process comprises a stage in which the components are dispersed Plasmatic in aqueous medium.

A second aspect of the present invention are the pharmaceutical compositions of pRNA associated with the components plasma obtainable by any of the processes defined in the first aspect or in any of its embodiments private individuals

A final aspect of the present invention is the use of pharmaceutical compositions according to any of the compositions of the second aspect for the preparation of a medicine.

Detailed description of the invention

The present inventors have discovered that, surprisingly, the plasma components, especially the plasma proteins and plasma lipids are capable of associate with pRNA, forming compounds, or complexes, that protect to the pRNA of the degradation of the RNases. For this it is vital that These plasma components are dispersed, or disaggregated. There is different techniques known in the art to make this dissociation, but one of those used, for its versatility, economy and ease is by using ultrasound, either applying the ultrasonic source directly in the solution or using a bathroom equipped with an ultrasound source. [Japanese Journal of Applied Physics, 47, 2008 pages 2000-2004].

The ratio between pRNA and the components Plasma can vary between 1: 100 and 1: 50,000 p / p, but preferably it varies between 1: 500 and 1: 10000. The proportions that they vary between 1: 3000 and 1: 5000 are preferred, because they are obtained high concentrations of pRNA, showing a good stability.

Preferably each of the chains of the pRNA duplex comprises between 15 and 40 nucleotides, referred to nucleotide pairs since it is a double stranded RNA for get into the cell and more preferably between 19 and 25 nucleotides

The dispersion can be carried out at different temperature ranges, but optimal temperatures they would be between 0 and 85 ° C, more preferably between 5 and 50 ° C, and even more preferably between 10 and 35 ° C. Regarding the duration of the stage Dispersion depends on the effectiveness of the method used, but normally, and especially when dispersed by ultrasound, this lasts at least 1 second, more preferably at least 15 seconds, even more preferably between 30 seconds and 2 hours, and even more preferably between 60 seconds and 30 min.

Dispersion, or disintegration, can be performed prior to mixing the plasma components with PARNI. Another alternative, which in turn is preferred, is the realization of the dispersion of plasma components in presence of pRNA, so the association is just after these have dispersed, so you avoid or / and reduces plasma components to be added again.

As already mentioned, the association between dispersed plasma components and pRNA are the ones that improve product stability. These plasma components can be obtained from different methods known in the art, such as lyophilization of blood plasma or ultrafiltration thereof, although it can be used as a source of plasma, plasma and / or serum components, directly without any previous treatment. . This is surprising because the pRNA is unstable in the plasma per se , but this pRNA shows a completely different behavior if the plasma has dispersed. This is useful because it facilitates the manufacturing process, since it is not necessary to isolate the plasma components.

The process of the invention can be used. for the manufacture of various types of formulations, but it have shown good absorption and biodistribution results when these are injectable.

Another advantage of the present process is that the improvement in absorption and biodistribution is not limited to any type of pRNA specifically. For example, it has been possible to form associated with pRNA without the need for it to be derivatized in some of its 3 'or 5' terminal positions, such as in the unmodified pRNA synthesized by chemical methods or synthesized to Starting from a template DNA by using RNA polymerase. Although it has been observed that stability can also be improved of pRNA when derivatized in its 3 'or 5' position with lipids, peptides or carbohydrates, or other organic molecule that retards the degradation of pRNA by nucleases, in any of its chains, either the guide or the companion. Also the chain antisense or sense may incorporate certain compounds to the improvement of the entry of the molecule into the cell, such as the introduction of lipophilic compounds such as cholesterol, acids fatty or etc.

The pRNA duplex can comprise derivatization such as substitutions selected from 2'-O-methyl ribonucleotide or 2'-deoxyribonucleotide or 2'-methoxyethyl ribonucleotide or 2'-fluoro-deoxyribonucleotide, or 2'-fluoro-arabinonucleotide, restricted conformation nucleosides (as known in English with the acronym LNA or HNA), RNA with phosphorothioate bonds, or Abbasic residues or ribitols or nucleosides containing bases modified such as 5-methylcytosine, 5-methyluracil, 4-alkynylcytosine, 5-alkynyluracil, 5-halogenocytosine, 5-halogenouracil, or other modified pyrimidines, 7-deazaadenine, 7-deazaguanine, hypoxanthine or other modified purines. Derivatization can incorporate any of the two chains, although the chain companion since it is more permissive to changes.

The pRNA duplex (Y) can inhibit and / or silence, being this inhibition / total silencing or partially with respect to a control, various genes of interest pharmacological such as tumor necrosis factor alpha (TNFα), Endothelial and vascular growth factor (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor factor (GM-CSF-1) and macrophages (M-CSF-1), angiopoietin (ANGPT), apolipoprotein (ApoB), vascular neovascularization (CNV), phosphoenol pyruvate carboxykinase (PEPCK), factor receptor human epidermal growth (HER2), inflammatory protein of macrophages (MIP2), receptor of N-methyl-D aspartate (NMDA), keratocyte-derived cytokine (KC), delta opium receptor (DOR), discoidin domain receptor (DDR1), gene of the PIV phosphoprotein (PIV-P), heme oxygenase (HMOX1), caveloina, dopamine transporter, green fluorescent protein and / or Swing sarcoma protein (EWS-FL / 1). The Preferred genes to silence / inhibit are the necrosis factor tumor tumor (TNFα), endothelial growth factor and vascular (VEGF), VEGF receptor (VEGF R1 / 2), suppressor factor of granulocyte colonies (GM-CSF-1) and macrophages (M-CSF-1). TNF-? Is an important mediator of the apoptosis both in inflammation and in the immune response. In addition, the overexpression of TNF-? Is confirmed in the triggering of the pathogenesis of many human diseases For all this the inhibition of this cytokine It is relevant from the biomedical point of view. PEPCK is a important protein in the gluconeogenesis process and is found overexpressed in diabetes. It is responsible for an increase in hepatic glucose production in diabetic patients and in models animals.

As examples of the present invention the pRNA sequence is: 1) anti-TNFα antisense or guide 5'-GAG GCU GAG ACA UAG GCA C-dT-dT-3 '(SEQ ID NO: 1) and 2) anti-TNF? Sense or companion: 5'-GUG CCU AUG UCU CAG CCU C-dT-dT-3 '(SEQ ID NO: 2). In capital letters the monomers of RNA, dT are indicated represent thymidine residues. This duplex of pRNA (anti TNF-?) Had already been previously described by efficiently regulate the TNF-? mRNA of mouse (D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766.). 12 have been prepared duplexes with various hydrophobic compounds at the ends either in the guide chain or in the accompanying chain (Table 1). Also 3) antisense chain or guide anti-PEPCK-C: 5'-UUACAUCUGGCUGAUUCUC-dT-dT-3 ' (SEQ ID NO: 5) and 4) Sense or companion chain anti-PEPCK-C: 5'-GAGAAUCAGCCAGAUGUAA-dT-dT-3 ' (SEQ ID NO: 6). In capital letters the monomers of RNA, dT represent thymidine residues.

The pharmaceutical compositions of the second aspect of the invention comprise a preferred embodiment of at less a pharmaceutically acceptable carrier or excipient. The excipients include any inert or non-active material used in the preparation of a pharmaceutical dosage form. By example, tablet excipients include, but are not limited to a: calcium phosphate, cellulose, starch or lactose. The forms of Liquid dosages also include oral liquids for example in the form of liquors or suspensions, as well as solutions injectable The pharmaceutical composition can be formulated for transdermal administration in the form of a patch. All compositions described above may optionally contain one or more of each of the following excipients: vehicles, diluents, dyes, flavoring agents, lubricants, agents solubilizers, disintegrants, binders and preservatives.

Although one of the advantages of this invention is that pRNA can be formulated without the need for a transfection agent, this can be added to the composition pharmaceutical to improve even if it fits the properties of this one, or to vectorize it. Preferred transfection agents are select from one or mixtures of the following compounds lipofectin, liptofectamine, oligofectamine, effectene, cellfectin, DOTAP, DOPE, fugene, polyethylene glycol, cholesterol, polyethyleneimide (PEI), Jet-polyethyleneimide, penetration peptides cellular, Trojan peptides, TAT peptide, penetratin, oligoarginine, poly-lysine, rabies virus glycoprotein, gold nanoparticles, dendrimers, carbon nanotubes, lipids cationic and liposomes.

The pharmaceutical compositions described in the present memory may have several uses, but preferred are for the treatment of cancer, inflammation, colitis, colitis ulcerative, Crohn's disease and / or rheumatoid arthritis. In In general, the compositions of the present invention are useful for the preparation of drugs for gene silencing.

Preferred pharmaceutical composition are for The present invention is presented in a form adapted to the oral or parenteral administration, preferably parenteral.

The administration of the compounds of this invention can be performed through any procedure that release the compound, preferably to the desired tissue. These procedures include oral, intravenous, intramuscular, subcutaneous or intramedullary, intraduodenal, etc. Preferably the Pharmaceutical composition of the present invention may administered locally by injection in an area near the region of interest (intramuscularly, subcutaneously, intradermally), injection in an area near the region of intravenous interest or injection.

For the purpose of administration parenteral, can be used as well as sterile aqueous solutions. Yes if necessary, such aqueous solutions can be buffered so adequate, and the liquid diluent first became isotonic with enough saline or glucose. These aqueous solutions They are especially suitable for injection purposes intravenous, intramuscular, subcutaneous and intraperitoneal. To this respect, all sterile aqueous media employed can be easily obtain by standard techniques well known for those skilled in the art.

Definitions

The blood plasma is the liquid component yellowing of the blood, in which blood cells should be normally suspended It represents 55% of the total volume blood The highest proportion is water (90% of the volume) and contains dissolved proteins, glucose, coagulation factors, ions minerals, hormones and carbon dioxide (plasma being the main means of transporting excreted products) and others components.

The blood plasma is prepared by centrifuging a tube of fresh blood, until the cells rush into the tube bottom. The blood plasma has an approximate density of 1025 kg / m3 or 1,025 kg / L. The blood serum is blood plasma without fibrinogen or other clotting factors.

Plasma contains a wide variety of proteins including albumin, immunoglobulins and proteins of coagulation as fibrinogen. Albumin constitutes about 60% of the total plasma proteins and is present in some concentrations between 35 and 55 mg / ml. Plasma is the main contributor of osmotic blood pressure and works as transport molecule of other molecules with low solubility aqueous such as lipid soluble hormones, enzymes, acids fatty, metal ions, pharmaceutical compounds. Albumin is structurally stable due to its 17 disulfide bridges and is the unique that has high water solubility and has the lowest isoelectric potential (pi) of plasma proteins. Because its structural integrity remains stable under conditions in which other proteins would be denatured.

The plasma is composed of 82.5% water, in addition to numerous inorganic and organic substances (solutes of plasma), distributed as follows: Plasma proteins (70%): fibrinogen (7%), immunoglobulins (38%), albumins (54%), other proteins (1%): VLDL, LDL, HDL, prothrombin, transferrin. Organic (non-electrolytic) metabolites and waste compounds (20%) phospholipids (280 mg / dL), cholesterol (150 mg / dL), triacylglycerols (125 mg / dL), glucose (100 mg / dL), urea (15 mg / dL), lactic acid (10 mg / dL), uric acid (3 mg / dL), creatinine (1.5 mg / dL), bilirubin (0.5 mg / dL) and bile salts (traces).

By pRNA it is understood in the context of the present invention as small fragments of interfering RNA double chain of synthetic or natural origin complementary to the sequence of genes that are used for specific inhibition of the genes of which they are complementary. The pRNAs are formed by two RNA chains that are called a guide chain (in English "guide strand" or "antisense strand") and string companion (in English "passenger strand" or "sense strand "). The pRNA guide chain joins a complex of proteins called RNA interference silencer complex (in English "RNA-interference silencing complex ", abbreviated as" RISC ") and the resulting complex is the person responsible for the cellular degradation of messenger RNA complementary to the guide chain. For the union of the guide chain to RISC complex is necessary to administer the double stranded RNA or pRNA since the guide chain alone cannot join the complex RISC

For pRNA associated with plasma components It is understood in the context of the present invention as the different complexes formed between pRNAs and molecules naturally present in the plasma formed after a process of disintegration or dispersion of plasma components. In the association process they take part in a majority electrostatic interactions and hydrophobic interactions with plasma components that are mostly blood proteins (albumins, fibrinogen, immunoglobulins, HDL, VLDL, LDL, prothrombin, transferrin etc ...) and secondly lipids naturally present in the plasma (triglycerides, cholesterol, phospholipids, bile acids, etc ...).

Plasma components are understood as molecules that are dissolved in the plasma naturally, and in especially the mixture of proteins and lipids present in the plasma. Due to the plasma composition this mixture may contain fibrinogen, immunoglobulins, albumins, and other proteins such as VLDL, LDL, HDL, prothrombin, transferine, etc ..., along with triglycerides, cholesterol, phospholipids, bile acids and etc. The proportions between plasma components for use in the present invention, and especially of proteins and lipids It can vary and can be adjusted as needed. These components they can basically consist of: either plasma proteins; or good plasma lipids; although they can also be found in Mixture form of protein ratios vs. lipids between 1: 1000 and 1000: 1. In a preferred embodiment the proportion of proteins to plasma lipids is around the relationship in the that is in the plasma.

By dispersing the plasma components in an aqueous medium is understood in the context of the present invention as the application of a physical force such as that produced by the action of ultrasound, to the solution comprising the plasma components, such as the plasma itself. , which results in the separation of molecular aggregates to give more or less isolated molecules exposing hydrophobic pockets and charged parts that were occluded by the aggregation process to the components of the aqueous solution, resulting in an increase in capacity of formation of electrostatic interactions and hydrophobic interactions with pRNAs. The degree of dispersion of plasma components could be estimated by various techniques that are based on the phenomenon
of the dispersion of the light by the biomolecules and that allow to estimate the hydrodynamic size of the biomolecules.

By the term "gene expression" is understood by the cellular production of a certain protein encoded by the corresponding gene. In general, the gene that It is found in the chromosomes present in the nucleus of cells it is transcribed generating a messenger RNA molecule that is released in the cytoplasm There the messenger RNA sequence directs the protein synthesis The result of this process, which is known as a gene expression, it is the synthesis of a protein whose sequence It is encoded by the corresponding gene.

Description of the figures

Figure 1 shows the in vivo inhibitory activity of several pRNAs conjugated with C 14 and C 18 lipids against mouse TNF-α, using protocols A and B in HeLa cells. 1. unmodified pRNA, 2. pRNA conjugated to cholesterol at the 3 'end of the accompanying chain, 7. pRNA conjugated to a C 14 lipid at the 3' end of the accompanying chain, 8. pRNA conjugated to a lipid C 18 at the 3 'end of the accompanying chain, 11. pRNA conjugated to a lipid C 14 at the 3' end of the accompanying chain and 2'OMe units, 12. pRNA conjugated to C 14 N at the 5 'end of the accompanying chain, 14. random sequence pRNA used as a negative control sequence. The description of sequences 1, 2, 7, 8, 11, 12 and 14 of the pRNAs is detailed in Table 1 and the chemical structure of the modifications is presented in Schemes 1 and 2, the data represent the means ±ES, n = 3 and are compared with the random sequence. *** p <0.001, * p <0.05. Statistical analysis was performed using the ANOVA method with the Bonferroni treatment.

Figure 2 shows the in vivo inhibitory activity of anti-TNF-? PRNA conjugated to acridine or quindoline, using protocols A and B in HeLa cells. 1. unmodified pRNA, 3. acridine conjugated pRNA at the 3 'end of the sense chain, 4. quindoline conjugated pRNA at the 3' end of the sense chain, 14. random sequence pRNA used as a negative control. The description of sequences 1, 3, 4 and 14 of the pRNAs is detailed in Table 1 and the chemical structure of the modifications is presented in Schemes 1 and 2, the data represent the means ±ES, n = 3 and are compared. With a random sequence. ** p <0.01, * p <0.05. Statistical analysis was performed using the ANOVA method with the Bonferroni treatment.

Figure 3 shows the effects of the formulation object of the invention on the in vivo inhibitory activity of several anti-TNF-? PRNAs conjugated with C14 and C18 lipids using protocols A and B in 4T1 cells. 1. unmodified pRNA, 2. pRNA conjugated with cholesterol at the 3 'end of the accompanying chain, 7. pRNA conjugated to a C 14 lipid at the 3' end of the accompanying chain, 8. pRNA conjugated to a lipid C 18 at the 3 'end of the accompanying chain, 9. pRNA conjugated to lipid C 14 at the 3' end of the guide chain, 10. pRNA conjugated to lipid C 18 at the 3 'end of the guide chain, 11. pRNA conjugated to a C 14 lipid at the 3 'end of the accompanying chain and 2'-OMe units, 12. pRNA conjugated to C 14 N at the 5' end of the accompanying chain , 14. random sequence pRNA used as a control sequence. The description of the sequences 1, 2, 7, 8, 9, 10, 11, 12 and 14 of the pRNAs is detailed in Table 1 and the chemical structure of the modifications is presented in Schemes 1 and 2, the data represent the means ± ES, n = 3 and are compared with a random sequence. ** p <0.001, ** p <0.01. Statistical analysis was performed using the ANOVA method with treatment with Bonferroni treatment.

Figure 4 shows the effect of the formulation object of the invention on the in vivo inhibitory activity of anti-TNF-? PRNA conjugated with acridine or quindoline, using protocols A and B in HeLa cells. 1. unmodified pRNA, 3. acridine conjugated pRNA at the 3 'end of the accompanying chain, 6. acridine conjugated pRNA at the 3' end of the guide chain, 4. quindoline conjugated pRNA at the 3 'end of the accompanying chain, 5. pRNA conjugated with quindoline at the 3 'end of the guide chain, 14. random sequence pRNA used as a negative control. The description of sequences 1, 3, 6, 4, 5 and 14 of the pRNAs is detailed in Table 1 and the chemical structure of the modifications is presented in Schemes 1 and 2, the data represent the means ±ES, n = 3 and are compared with the random sequence. ** p <0.01, * p <0.05. Statistical analysis was performed using the ANOVA method with the Bonferroni treatment.

Figure 5A shows the effects of the pRNA duplex in the absence of serum and Figure 5B shows the effects of the formulation object of the invention on the in vivo inhibitory activity of pRNA against PEPCK in FAO cells. A. unmodified pRNA, B. pRNA conjugated with cholesterol at the 3 'end of the accompanying chain, 14. random sequence pRNA used as a negative control. The description of the A, B and 14 sequences of the pRNAs is detailed in Table 1 and the chemical structure of the modifications is presented in Schemes 1 and 2. The amounts of mRNA produced by the cells were determined after 48 hours of treatment. with the pRNAs. The data represent the means ±ES, n = 3 and are compared with a random sequence. ** p <0.01, * p <0.05. Statistical analysis was performed using the ANOVA method with the Bonferroni treatment.

Figure 6 shows the fluorescence measurement of the siGLO pRNA in 4T1 cells by flow cytometry. 4T1 cells were transfected with 100 nM siRNA pRNA (Cy3) incubated with different amounts of fetal bovine serum (0 µL to 25 µL) using ultrasound with the protocol that we have called the formulation object of the invention. After 24 hours of incubation, the cells were analyzed in a flow cytometer. The cellular input of the Cy3-labeled pRNA was quantified by the measurement of the relative fluorescence signal in the Cy3 channel. The cells without pRNA were used as a fluorescence control. The data represent means ± NES, n = 3 and are compared with cells transfected with the pRNA without FBS. *** p <0.001, ** p <0.01. ANOVA test, Bonferroni post-test.

Figure 7 shows the biodistribution of a pRNA fluorescent.

Five mice of the ICR strain of 8 weeks of age were used. 2 mice were injected iv with 10 µg of siRNA pRNA according to the formulation object of the invention with 450 µL of mouse serum, another 2 mice received IV of the siRNA siRNA dissolved directly in 450 µL of physiological serum and 1 mouse that was not injected, was used as a fluorescence control. After 4 hours, the animals were sacrificed and the organs perfused in 4% PFA and then cut the tissues and observe them under the confocal microscope. The data represent the means ± S, n = 3 stacks of photos of each tissue and the formulation object of the invention is compared with the siRNA pRNA dissolved directly in the serum, between each of the tissues. *** p <0.001, ** p <0.01, * p <0.05 ANOVA test, Bonferroni post-test.

Figure 8 shows a process diagram for the formulation of the dispersion of pRNA and serum components by using sonication.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples Oligonucleotide synthesis

The oligoribonucleotides were prepared using a synthesizer from the Applied Biosystems Model 3400 trading house using 2-cyanoethyl phosphoramidites and tert-butyldimethylsilyl (TBDMS) type protectors for the protection of the 2 'position hydroxyl group. The following solutions were used: 0.4 M 1H-tetrazole in ACN (catalyst); 3% trichloroacetic acid in DCM (detritilation), acetic anhydride / pyridine / tetrahydrofuran (1: 1: 8) (capping A), 10% N-methylimidazole in tetrahydrofuran (capping B), 0.01 M iodine in tetrahydrofuran / pyridine / water (7: 2: 1) (oxidation). In the RNA sequences, the last DMT was removed since the DMT group is not completely stable to fluoride treatment. The average yield per stage of addition of a nucleotide was about 97-98% for RNA monomers. The polymeric supports obtained were treated with a concentrated solution of ammonia-ethanol (3: 1) for 1 h at 55 ° C. The supports were washed with ethanol and the resulting solutions were combined and evaporated to dryness. The resulting products were treated with 0.15 ml of triethylaminetris (hydrofluoride) / triethylamine / N-methylpyrrolidone (4: 3: 6) for 2.5 h at 65 ° C in order to eliminate TBDMS groups. The reactions were stopped by the addition of 0.3 ml of isopropoxytrimethylsilane and 0.75 ml of ether. The resulting mixtures were stirred and cooled to 4 ° C. A precipitate formed which was centrifuged at 7000 rpm for 5 min at 4 ° C. The precipitates were washed with ether and centrifuged again. The residues were dissolved in water and the conjugates were purified by HPLC. Column: Nucleosil 120-10 C18 (250x4 mm); a linear gradient of 20 min from 0% to 50% B was used; with a flow of 3 ml / min. The composition of the solutions used in the HPLC were: solution A: 5% acetonitrile (ACN) in 100 mM triethylammonium acetate (pH 6.5) and solution B: 70% ACN in 100 mM triethylammonium acetate pH 6.5. The purified products were analyzed by mass spectrometry
MALDI-TOF.

The spectra of MALDI-TOF are performed on a Perseptive Voyager DETMRP mass spectrometer, equipped with a 337 nm nitrogen laser using a pulse of 3ns. The matrix used contained 2,4,6-trihydroxyacetophenone (THAP, 10 mg / ml in ACN / water 1: 1) and ammonium citrate (50 mg / ml in water).

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Oligonucleotides

The following RNA sequences were obtained from commercial sources (Sigma-Proligo, Dharmacon): 5'-CAG UCG CGU negative control companion chain UUG CGA CUG G-dT-dT-3 '(SEQ ID NO: 3), 5'-CCA GUC GCA negative control guide chain AAC GCG ACU G-dT-dT-3 '(SEQ ID NO: 4), anti-TNF? Guide chain: SEQ ID NO: 1 and anti-TNFα companion chain: SEQ ID NO: 2. RNA monomers are detailed in capital letters, the abbreviation dT corresponds thymidine. The anti sense chain TNF-? With cholesterol at the 3 'end: SEQ ID NO: 1-cholesterol was prepared using the support commercial cholesterol-tetraethylene glycol (TEG) -3'-CPG (Glen Research). He fluorescent pRNA labeled with Cy3 was obtained from sources commercial (siGLO Thermo scientific, Dharmacon USA). The sequences of anti TNF-? pRNA had been described pRNA in the literature to inhibit the mRNA of Mouse TNF-? [D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766.].

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Cell cultures, transfection and cell assays

HeLa cells were cultured under usual conditions: 37 ° C, 5% CO 2, modified Dulbecco Eagle medium, 10% fetal bovine serum, 2 mM L-glutamine, supplemented with penicillin (100 U / ml) and streptomycin (100 mg / mL) All experiments were done at a confluence of 40-60%. HeLa cells were transfected with 250 ng of the plasmid expressing the mouse TNF-α gene using lipofectin (Invitrogen) and following the instructions of the
maker.

4T1 mouse cells were cultured in usual conditions. 100 nM of each of the pRNA duplexes incubated with 10% fetal bovine serum (FBS) before adding to 4T1 cells. After 24 hours the amount of TNF-? Produced by the cells was analyzed by the ELISA test.

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Practical example of the method object of the invention

i) The pRNAs with general formula (N19TT / N19TT) were added to the fetal bovine serum at a rate of 0.33? pRNA / 25 µl of serum.

ii) The pRNA / serum complex was placed in a ultrasonic bath for 5-10 minutes.

iii) The pRNA / serum complex treated with Ultrasound was added to the cell culture in the presence of medium fresh cell culture.

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1) Synthesis of oligoribonucleotides containing hydrophobic molecules for use in interfering RNA assay .

The following genes were chosen as the target genes genes that code for tumor necrosis factor (TNF-?): 1) anti-TNF? guide chain SEQ ID NO: 1 and 2) anti-TNF? accompanying chain SEQ ID NO: 2. This duplex of pRNA (anti TNF-?) Had already been previously described by efficiently regulate the TNF-? mRNA of mouse (S D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766.). 12 have been prepared duplexes with various hydrophobic compounds at the ends either in the guide chain or in the accompanying chain (Table 1).

The following sequences were selected as genes encoding PEPCK-C. 1) chain antisense or anti-PEPCK-C guide: SEQ ID NO: 5 and 2) Sense or companion chain anti-PEPCK-C: SEQ ID NO: 6. You are sequences have not been previously described.

Seven sequences of oligonucleotides conjugated with C 14 and C 18 derivatives: Two guide and five companion chains, conjugated with C 14 and C 18 at the 3 'end (see scheme 1 and Table 1).

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Scheme one

Chemical structure of lipids (C 14, C 18 and C 14 N) attached to the parni

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one

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The oligonucleotides corresponding to the Companion chain 7 and 8 have derivatives C 14 and C 18 in the 3 'end respectively. Oligonucleotide 12 has a derivative of C 14 N at the 5 'end (table 1). Oligonucleotide 11 has a C_ {14} derivative at the 3 'end and several units 2'-O-methyl along the chain less companion in the last two thymidines. It has been described that 2'-O-methyl units stabilize RNA chains of nuclease degradation. The position of 2'-OMe units (mC = 2'-O-methyl-C and mU = 2'-O-methyl-U) in the oligonucleotide 11 is 5'-GUG C (mC) U AUG (mU) (mC) U (mC) AG (mC) C (mU) CTT-3 '(SEQ ID NO: 7) -C_ {14}.

Finally, four were prepared oligonucleotides conjugated with acridine or quindoline at the end 3 '(table 1): Two accompanying chains conjugated with acridine 3 or quindoline 4 respectively and two guide chains conjugated with acridine 6 or quindoline 5. The guide chain conjugated with cholesterol at the 3 'end it was prepared using commercial reagents. The RNA sequence synthesis has been developed by the inventors of the patent.

TABLE 1 Sequence of pRNAs containing molecules hydrophobic

2

3

G: Chain guide / A: companion chain. The nucleotide sequences of said chains are presented from 5 'to 3'. mC and mU represent the position of derivatives 2'-O-methyl-C and 2'-O-methyl-U respectively.

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Scheme 2

Chemical structure of acridine and quindoline modifications included in one of the 3'-terminal positions of the parni

4

2) Inhibition of TNF-?. The guide and companion chains were hybridized and the result was a duplex or pRNA that was used to inhibit the expression of the TNF-α gene. First, the inhibitory properties of pRNA in HeLa cells were evaluated with and without the formulation object of the invention. These cells do not express the mouse TNF-α gene therefore, a cotransfection was performed with the plasmid pCAm containing the mouse TNF-α gene. Two different protocols were studied.

Protocol A) HeLa cells were transfected with 250 ng pCAm mouse plasmid TNF-? Using lipofectin. One hour later the cells were treated with the pRNA (100 nM) without using any transfection reagent, but were incubated with fetal serum cattle and treated with ultrasound before the process of transfection After 48 hours the amount of TNF-? Produced by the cells was analyzed by ELISA.

Protocol B) HeLa cells were transfected with 250 ng of the mouse plasmid pCAm TNF-? Using lipofectin as described in section A. An hour later the cells were treated with the pRNA duplex (100 nM) in the absence of serum. After 48 hours the amount of TNF-? produced by the Cells were analyzed by ELISA.

The results of this experiment show that the amount of TNF-? produced after 48 hours of the addition of modified pRNA duplexes incubated with 10% serum and ultrasound (A) or after addition without the incubation (B), are clearly different. An inhibition was observed between 40 and 50% in protein production TNF-? Compared to the sequence pRNA randomized, only when the pRNA was transfected using the formulation object of the invention. The presence of the units of lipids at the 3 'and 5' ends of the sense chain increased TNF-? expression inhibition.

Figure 2 indicates that the pRNAs conjugated with acridine or quindoline pretreated with serum and ultrasound produced an inhibition between 30 and 50% in the production of TNF-? Comparing with the randomized pRNA used as a negative control No inhibition was observed in the absence of formulation object of the invention. Only quindoline in the 3 'end of the sense chain increased the inhibition of the TNF-? expression.

The properties were studied below. inhibitors of pRNA in 4T1 mouse cells. These cells produce mouse TNF-? endogenously. In these experiments the cells were treated with the pRNA duplex (100 nM) without the use of any transfection reagent. The pRNA were incubated with serum and ultrasound or simply added to cells without prior treatment. At 24 hours the amount of TNF-? produced by the cells It was analyzed by ELISA.

The results of the inhibition of TNF-? By lipid conjugated pRNAs are shown in figure 3. As described for HeLa cells, it can observe a strong inhibition in the production of TNF-? Only when the formulation was used object of the invention. The highest inhibition (80%) was observed when the pRNA duplex conjugated to the lipid was used C 14 at the 3 'end of the accompanying chain (7).

The results of the inhibition of TNF-? By acridine-conjugated pRNAs or Quindoline are shown in Figure 4. The guide chains and Companion were modified at the 3 'end (3 and 6). As it can observe, a strong inhibition in production was found of TNF-? using the formulation object of the invention. The highest inhibition (60%) was observed with the pRNA conjugated with quindoline at the 3 'end of the accompanying chain (4).

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3) Inhibition of PEPCK-C .

The guide and companion sequences complementary to PEPCK were hybridized and the resulting duplex was used to inhibit the expression of the PEPCK-C gene. Be studied the properties of pRNA in FAO cells (hepatoma of rat) with and without the formulation object of the invention. These cells express the PEPCK-C gene endogenously (pckl) so so much it was not necessary to carry out cotransfection with the plasmid correspondent. The following protocols were used:

A) FAO cells were treated with the duplex of pRNA (100 nM) in the absence of serum. After 48 hours the amount of pepCK-C mRNA produced by Cells were analyzed by qRT-PCR.

B) FAO cells were treated with the duplex of pRNA (100 nM) incubated with fetal bovine serum and ultrasound. After 48 hours the amount of PEPCK-C mRNA produced by the cells was analyzed by qRT-PCR.

The results of this experiment demonstrate that the pRNA incubated with serum and ultrasound improves significantly the gene silencing of the target gene Pck1 in FAO cells. It is interesting to note that the improvement of entry of the pRNA by action of the formulation object of the invention does not require modifications to the pRNA to help Increase efficiency Using this methodology the pRNA without modify is a good mRNA inhibitor of PEPCK-C.

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4) Cellular entry of a pRNA labeled with a fluorescent molecule (Cy3) using the formulation object of the invention. ''

The method was optimized, assuming that the The efficiency of the method depended on the interaction of the pRNA with the plasma components, determining the best proportion of pRNA / serum. To do this, 4T1 mouse cells were transfected with a fluorescent pRNA labeled with Cy3 previously incubated with different volumes of fetal bovine serum and ultrasound (from 0 µL to 25 µL), and we measured the entry of pRNA into the cells by flow cytometry (DakoCytomation, MoFlo model) at a 1000 cell rate per second.

The images obtained using the flow cytometer, show that when the siRNA pRNA was transfected directly on the cells with medium supplemented with serum, that is, without the formulation object of the invention, only 20% of the cells in the field were transfected, while in the case of the formulation object of the invention of the siRNA pRNA with 15 µl of FBS, 50% of the cells exhibited a great fluorescence (Figure 6). According to these data, the most efficient proportion to ensure a better entry of pRNA into cells is 0.33 µg pRNA / 15 µL serum which will be the concentration used in the in vivo assays.

It was also observed that as the volume of serum, the efficiency of entry of pRNA into cells decreases, this may be due to a possible saturation of the sites of union that have the proteins present in the serum.

In vivo testing

5) In vivo administration of the siRNA pRNA in mice .

5 mice of the 8-week ICR strain were used of age (Harían, Iberian Interface Spain). The animals were housed in standard environmental conditions, temperature 22ºC, relative humidity of 70-80% and with a cycle of lighting of 12 hours a day, until the moment of making the essays.

All protocols used were approved previously by the ethical committee of the University of Barcelona and the Animal handling was governed by the regulations of the European Community.

The trial consisted of administering by injection in the tail vein of mice, 450 µL of a solution of Cy3 fluorescent pRNA, incubated with ultrasound or not with serum mouse preserving a ratio of 0.33 µg of pRNA: 15 µL of serum After 4 hours, the animals were sacrificed with ether and tissues were perfused with 4% PFA. Then the tissues were cut in a Criostat (Leica) with a size of 30 ? to be mounted on slides and observed under the confocal microscope (Leica model. TCS NT). 3 photos were taken of Longitudinal cuts of each of the organs, keeping the same acquisition parameters between organs. Fluorescence observed in the photos, it was quantified using a program of computer, the MacBiophotonics ImageJ that allows to obtain values Quantitative tissue fluorescence.

As shown in figure 7, after 4 hours of administration via i.v., the levels of SiGLO fluorescence without the formulation object of the invention were lower in all organs, while they were detected significant levels between 20 and 50% more lung pRNA, heart, liver, spleen and adipose tissue when it was pre-incubated with mouse serum Indicating this that the formulation object of the invention improves the biodistribution properties of PARNI.

<110> Higher Research Council Scientific (CSIC)

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University of Barcelona

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Claims (31)

1. A process for obtaining a formulation comprising pRNA associated with plasma components, characterized in that the process comprises a stage in which the plasma components are dispersed in aqueous medium. 2. The process according to the preceding claim, characterized in that the dispersion of the plasma components is carried out by ultrasound. 3. The process according to any of the preceding claims, characterized in that the ratio between pRNA and plasma components varies between 1: 100 and 1: 50,000. 4. The process according to the preceding claim, characterized in that the ratio between pRNA and plasma components varies between 1: 500 and 1: 10000. 5. The process according to the preceding claim, characterized in that the ratio between pRNA and plasma components varies between 1: 3000 and 1: 5000. 6. The process according to any of the preceding claims, characterized in that plasma and / or blood serum is used as the source of the plasma components. 7. The process according to any of the preceding claims, characterized in that the formulation is injectable. 8. The process according to any of the preceding claims, characterized in that the pRNA is not derivatized at its 3 'or 5' end in any of its chains. 9. The process according to the preceding claim, characterized in that the pRNA is from a natural source (isolated from cells), or obtained by enzymatic copying of a DNA template by using RNA polymerase or synthesized from a template DNA by the use of RNA polymerase. 10. The process according to any of claims 1 to 7, characterized in that the pRNA is derivatized at its 3 'or 5' position with at least one lipid, at least one peptide, at least one carbohydrate or any combination thereof. 11. The process according to any of the preceding claims, characterized in that the pRNA comprises between 15 and 40 nucleotides. 12. The process according to any of the preceding claims, characterized in that the pRNA comprises between 19 and 25 nucleotides. 13. The process according to any of the preceding claims, characterized in that the dispersion is carried out at a temperature between 0 and 85 ° C. 14. The process according to the preceding claim characterized in that the dispersion is carried out at a temperature between 5 and 50 ° C. 15. The process according to the preceding claim, characterized in that the dispersion is carried out at a temperature between 10 and 35 ° C. 16. The process according to any of the preceding claims, characterized in that the dispersion is carried out for at least 1 second. 17. The process according to the preceding claim, characterized in that the dispersion is carried out for at least 15 seconds. 18. The process according to the preceding claim, characterized in that the dispersion is carried out for at least 30 seconds and 2 hours. 19. The process according to the preceding claim, characterized in that the dispersion is carried out at least 60 seconds and 30 min. 20. The process according to any of the preceding claims, characterized in that pRNA duplex comprises at least one substitution selected from 2'-O-methyl ribonucleotide; 2'-deoxyribonucleotide;2'-methoxyethylribonucleotide;2'-fluoro-deoxyribonucleotide;2'-fluoro-arabinonucleotide; restricted conformation nucleosides; RNA with phosphorothioate bonds; Abbasic residues or ribitols; or nucleosides containing modified bases such as 5-methylcytosine, 5-methyluracil, 4-alkynylcytosine, 5-alkynyluracil, 5-halogenocytosine, 5-halogenouracil, 7-deazaadenine, 7-deazaguanine or hypoxanthine. 21. The process according to any one of the preceding claims, characterized in that the pRNA duplex partially / totally inhibits / silences the expression of at least one of the following genes: tumor necrosis factor alpha (TNFα), endothelial growth factor and vascular (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor (GM-CSF-1) and macrophage (M-CSF-1), angiopoietin (ANGPT), apolipoprotein (ApoB), neovascularization vascular (CNV), carboxy kinase phosphoenol pyruvate (PEPCK), human epidermal growth factor receptor (HER2), macrophage inflammatory protein (MIP2), N-methyl-D aspartate (NMDA) receptor, keratocyte-derived cytokine (KC) ), delta opium receptor (DOR), Discoidine domain receptor (DDR1), PIV phosphoprotein gene (PIV-P), heme oxygenase (HMOX1), caveloin, dopamine transporter, green fluorescent protein and sarcoma protein from Swing (EWS-FL / 1). 22. The process according to the preceding claim, characterized in that the duplex of pRNA (Y) inhibits / silences, totally or partially, the expression of at least one of the following genes: tumor necrosis factor alpha (TNFα), factor of Endothelial and vascular growth (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor factor (GM-CSF-1) and macrophage (M-CSF-1). 23. The process according to any of the preceding claims, characterized in that the pRNA sequence is selected from: anti-TNF? Antisense or guide 5'SEQ ID NO: 1; anti-TNF? sense or companion: SEQ ID NO: 2; antisense chain or anti-PEPCK-C guide: SEQ ID NO: 5; SEQ ID NO: 6. 24. A pharmaceutical composition of pRNA associated with plasma components obtainable by any of the processes defined in the preceding claims. 25. The pharmaceutical composition according to the preceding claim, characterized in that it comprises at least one pharmaceutically acceptable excipient or vehicle. 26. The pharmaceutical composition according to the preceding claim, characterized in that it comprises at least one transfection agent. 27. The pharmaceutical composition according to the preceding claim, characterized in that the transfection agent is selected from the list comprising the following compounds: lipofectin, liptopofectamine, oligofectamine, effectene, cellfectin, DOTAP, DOPE, fugene, polyethylene glycol, cholesterol, polyethyleneimide (PEI) ), Jet-polyethyleneimide, cell penetration peptides, Trojan peptides, TAT peptide, penetratin, oligoarginine, poly-lysine, rabies virus glycoprotein, gold nanoparticles, dendrimers, carbon nanotubes, cationic lipids and liposomes. 28. The pharmaceutical composition according to any of claims 24 to 27, wherein it is presented in a form adapted to oral or parenteral administration, preferably parenteral 29. The use of pharmaceutical compositions according to any of the five preceding claims for the Preparation of a medicine. 30. The use according to the preceding claim for the preparation of a drug for gene silencing. 31. Use according to either of the two previous claims for the preparation of a medicament for the treatment of cancer, inflammation, colitis, colitis ulcerative, Crohn's disease or rheumatoid arthritis.